ES2375288T3 - PROCEDURE TO DETERMINE ANALYTE CONCENTRATIONS CORRECTED WITH HEMATOCRITE. - Google Patents

Abstract

Description is provided herein for an embodiment of a method determining a hematocrit-corrected glucose concentration. The exemplary method includes providing a test strip having a reference electrode and a working electrode, wherein the working electrode includes a plurality of microelectrodes and is coated with at least an enzyme and a mediator. The method can be achieved by: providing a test strip comprising a reference electrode and a working electrode formed with a plurality of microelectrodes and coated with a reagent layer; applying a fluid sample to the test strip for a reaction period; applying a test voltage between the reference electrode and the working electrode; measuring a test current as a function of time; measuring a steady state current value when the test current has reached an equilibrium; calculating a ratio of the test current to the steady state current value; plotting the ratio of the test current to the steady state current value as a function of the inverse square root of time; calculating an effective diffusion coefficient from the slope of the linearly regressed plot of the ratio of the test current to the steady state current value as a function of the inverse square root of time; and calculating a hematocrit-corrected concentration of analyte.

Description

Procedure for determining analyte concentrations corrected with hematocrit

1. Priority

The present application claims the benefits of the priority under Article 119, title 35 of the U. S. C. from the provisional application S. N. 60 / 850.173 filed on October 5, 2006, entitled: "Procedure for determining analyte concentrations corrected with hematocrit".

2. Background of the invention

Electrochemical glucose test strips, such as those used in the OneTouch® Ultra® complete blood test kit, which is available through LifeScan, Inc., are designed to measure the concentration of glucose in a sample of blood of patients with diabetes. The glucose measurement is based on the specific oxidation of glucose by the flavo-glucose oxidase enzyme. The reactions that can take place in a glucose test strip are summarized below in equations 1 and 2.

D – Glucose + GO (ox) � Gluconic acid + GO (red) (1)

GO (network) + 2 FE (CN) 6 3– � GO (ox) + 2 Fe (CN) 6 4– (2)

As shown in equation 1, glucose is oxidized in gluconic acid by the oxidized form of glucose oxidase (GO (ox)). It should be noted that GO (ox) can also be referred to as an "oxidized enzyme." During the reaction in equation 1, the oxidized enzyme GO (ox) becomes its reduced state, which is indicated as GO (red) (ie, "reduced enzyme"). Then, the reduced enzyme GO (red) is re-oxidized back to GO (ox) by a reaction with Fe (CN) 6 3– (referred to as either the oxidized mediator or ferricyanide) such as is shown in equation 2. During the regeneration of GO (network) back to its oxidized state GO (ox), Fe (CN) 6 3– is reduced to Fe (CN) 6 4– (referred to as either the reduced mediator or ferrocyanide).

When the reactions described above are carried out by applying a test voltage between two electrodes, a test current can be created by electrochemical reoxidation of the reduced mediator on the electrode surface. Therefore, because, in an ideal environment, the amount of ferrocyanide that is created during the chemical reaction described above is directly proportional to the amount of glucose in the sample that is placed between the electrodes, the test current generated would be proportional to the glucose content of the sample. A mediator, such as ferricyanide, is a compound that accepts electrons from an enzyme such as glucose oxidase and then donates the electrons to an electrode. As the concentration of glucose in the sample increases, the amount of reduced mediator formed also increases, thus, there is a direct relationship between the test current resulting from the reoxidation of the reduced mediator and the glucose concentration. In particular, the transfer of electrons through the electrical contact surface results in a test current flow (2 moles of electrons per mole of glucose that is oxidized). Reference may be made to the test stream that results from the introduction of glucose, therefore, as a glucose stream.

Because it can be very important to know the concentration of blood glucose, particularly in people with diabetes, test meters have been developed using the principles outlined above to allow the average person to sample and test their blood. to determine the concentration of glucose at any given time. The generated glucose current is monitored by the test meter and converted to a glucose concentration reading using an algorithm that relates the test current to a glucose concentration through a simple mathematical formula. In general, test meters work in conjunction with a disposable test strip that includes a sample receiving chamber and at least two electrodes that are arranged inside the sample receiving chamber in addition to the enzyme (for example, glucose oxidase) and the mediator (for example, ferricyanide). During use, the user punctures his finger or other convenient place to induce bleeding and introduces a blood sample into the sample reception chamber, thereby initiating the chemical reaction set forth above.

In electrochemical terms, the function of the meter is twofold. First, it provides a bias voltage (approximately 0.4 V in the case of OneTouch® Ultra®) that polarizes the electrical contact surface and allows current flow at the carbon electrode of the work surface. Second, it measures the current flowing in the external circuit between the anode (the working electrode) and the cathode (the reference electrode). It can therefore be considered that the test meter is a simple electrochemical system that operates in a two electrode mode although, in practice, third and even fourth electrodes can be used to facilitate the measurement of glucose and / or perform other functions on the meter.

In most situations, the equation described above is considered to be a sufficient approximation of the chemical reaction that takes place in the test strip and that the test meter emits

as output a sufficiently accurate representation of the glucose content of the blood sample. However, in certain circumstances and for certain purposes, it may be advantageous to improve the measurement accuracy. For example, blood samples that have a high level of hematocrit or a low level of hematocrit may cause a glucose measurement to be inaccurate.

A hematocrit level represents a percentage of the volume of a whole blood sample occupied by red blood cells. The hematocrit level can also be represented as a fraction of red blood cells present in a whole blood sample. In general, a blood sample with a high hematocrit content is more viscous (up to about 10 centipoise at 70% hematocrit) than a blood sample with a low hematocrit content (approximately 3 centipoise at 20% hematocrit). In addition, a blood sample with a high hematocrit content has a higher oxygen content than a blood with a low hematocrit content due to the concomitant increase in hemoglobin, than a carrier for oxygen. Therefore, the level of hematocrit can influence the viscosity and oxygen content of the blood. As will be described later, both the viscosity and the oxygen content can change the magnitude of the glucose stream and in turn lead to a low concentration of glucose.

A high viscosity sample (i.e. a blood sample with high hematocrit content) may result in the test current decreasing for a variety of factors such as a decrease in 1) the rate of dissolution of the enzyme and / or the mediator, 2) the reaction rate of the enzyme, and 3) the diffusion of a reduced mediator to the working electrode. A decrease in current that is not based on a decrease in glucose concentration can potentially result in an inaccurate glucose concentration being measured.

A slower dissolution rate of the reagent layer can slow down the enzymatic reaction as shown in equations 1 and 2 because the oxidized enzyme GO (ox) must first dissolve before it can react with the glucose. Similarly, ferricyanide (Fe (CN) 6 3–) must first dissolve before it can react with the reduced enzyme GO (red). If the undissolved oxidized GO (ox) enzyme cannot oxidize glucose, then the reduced GO (red) enzyme cannot produce the reduced mediator Fe (CN) 6 4– which is necessary to generate the test current. In addition, the oxidized enzyme GO (ox) will react with glucose and the oxidized mediator Fe (CN) 6 3– more slowly if it is in a high viscosity sample as opposed to a low viscosity sample. The slowest reaction rate with high viscosity samples is attributed to a global decrease in mass diffusion. Both the oxidized enzyme GO (ox) and glucose must collide and interact with each other so that the reaction takes place as shown in equation 1. The ability of the oxidized enzyme GO (ox) and glucose to collide and interact with each other It slows down when they meet a viscous sample. Even more, the reduced mediator Fe (CN) 6 4– will diffuse to the working electrode more slowly when dissolved in a high viscosity sample. Because the test current is typically limited by the diffusion of the reduced mediator Fe (CN) 6 4– in the working electrode, a high viscosity sample will also attenuate the test current. In summary, there are several factors that cause the test current to decrease when the sample has an increased viscosity.

A high oxygen content can also lead to a decrease in the test current. The reduced enzyme (GO (red)) can reduce oxygen (O2) to hydrogen peroxide as shown in equation 3.

GO (network) + O2 � GO (ox) + H2O2 (3)

As indicated above, the reduced enzyme GO (red) can reduce to ferrocyanide (Fe (CN) 6 4–) also ferricyanide (Fe (CN) 6 3-) as shown in equation 2. Therefore , oxygen can compete with ferricyanide to react with the reduced enzyme (GO (red)). In other words, the occurrence of the reaction in equation 3 will probably result in a decrease in the speed of the reaction in equation 2. Due to such a competition between ferricyanide and oxygen, a higher oxygen content will result in less ferrocyanide. In turn, a decrease in ferrocyanide would result in a decrease in the magnitude of the test current. Therefore, a high oxygen content of blood sample can potentially decrease the test current and affect the accuracy of glucose measurement.

As such, applicants of the present invention have a great interest in the development of procedures that reduce the effects of hematocrit on a glucose measurement. In certain protocols, a previously filtered filtered blood membrane that is separated from the reagent layer has been used to remove red blood cells and thereby reduce the effect of hematocrit. The pre-cast blood filtering membrane that is separated from the reagent layer can be disposed on the working electrode. The use of a previously discrete cast blood filtration membrane is unsatisfactory in that it requires a more complex test strip, an increased sample volume, and an increased test time. The blood filtering membrane retains a certain amount of blood that does not come in contact with the working electrodes which results in a need for a larger blood sample. In addition, a finite amount of time is needed for blood to seep through the membrane resulting in an increase in global test times. Therefore, applicants of the present invention recognize that it would be advantageous to reduce the effects of hematocrit without using a pre-cast blood filtering membrane that is separated from the reagent layer.

In the prior art, the effect of hematocrit can be reduced by applying multiple test voltages such as, for example, a sinusoidal test voltage. However, applying a sinusoidal test voltage results in a more complex and expensive test meter. In addition, the test meter needs to measure the test current accurately and accurately at predetermined time intervals. Electronic components can be expensive and complicated for a test meter to accurately and accurately apply multiple test voltages.

Applicants of the present invention noted that it would be advantageous to implement a system that had a test meter that applied only a test voltage and a test strip that did not use a pre-cast membrane to reduce the effects of hematocrit. The system uses instead a test strip that has a working electrode with a plurality of microelectrodes formed thereon. More particularly, the applicants of the present invention recognize that it would be advantageous to develop an algorithm that mathematically processes the collected test current using a test voltage such that an accurate concentration of glucose that reduces the effects of hematocrit can be determined.

Additionally, applicants of the present invention have determined that it would be beneficial to provide a mechanism by which the test meter can differentiate between a body fluid, for example whole blood, and a control solution. Similarly, it would be beneficial to provide a method by which a test meter can determine whether a test strip includes a plurality of microelectrodes formed in a working electrode.

Summary of the invention

In one aspect, a procedure is provided to determine a glucose concentration corrected with hematocrit. The exemplary method includes providing a test strip having a reference electrode and a working electrode, in which the working electrode includes a plurality of microelectrodes and is coated with at least one enzyme and a mediator. The process can be carried out: by providing a test strip comprising a reference electrode and a working electrode formed with a plurality of microelectrodes and coated with a reagent layer; applying a sample of fluid to the test strip during a reaction period; applying a test voltage between the reference electrode and the working electrode; measuring a test current as a function of time; measuring a steady state current value when the test current has reached equilibrium; calculating a proportion of the test current with respect to the steady state current value; plotting the proportion of the test current with respect to the steady state current value as a function of the inverse of the square root of time; calculating an effective diffusion coefficient from the slope of the linear regression graphical representation of the proportion of the test current with respect to the steady state current value as a function of the inverse of the square root of time; and calculating a corrected concentration with analyte hematocrit.

In another aspect of the present invention, the exemplary method further includes steps to distinguish between a body fluid and a control solution. The procedure includes comparing the calculated value of the effective diffusion coefficient with respect to an acceptance range for either a body fluid or a control solution, depending on the sample applied to the test strip. If the calculated value is not within the acceptance range for body fluid or control solution, the test meter will not allow the user to proceed with the tests and will display an appropriate error message.

In another aspect, a method of determining a type of fluid sample applied to the test strip is provided. The procedure can be carried out: by providing a test strip having a reference electrode and a working electrode, in which the working electrode is formed with a plurality of microelectrodes and is coated with a reagent layer; applying a sample of fluid to the test strip during a reaction period; applying a test voltage between the reference electrode and the working electrode; measuring a test current as a function of time; measuring a steady state current value when the test current has reached equilibrium; calculating a proportion of the test current with respect to the steady state current value; plotting the proportion of the test current with respect to the steady state current value as a function of the inverse of the square root of time; calculating an effective diffusion coefficient from the slope of the linear regression graphical representation of the proportion of the test current with respect to the steady state current value as a function of the inverse of the square root of time; determining a type of a fluid sample applied to the test strip by comparing a measured value for the effective diffusion coefficient versus an acceptance range for a body fluid and a control solution; and displaying an appropriate error message if the effective diffusion coefficient does not pass the acceptance interval for the type of fluid sample applied to the test strip.

In yet another aspect of the present invention, the exemplary method further includes steps to determine if a test strip includes a microelectrode arrangement. The procedure includes using the effective diffusion coefficient to calculate an effective diffusion coefficient corrected in temperature. The calculated value for the effective diffusion coefficient corrected in temperature is then compared with an acceptance range for a test strip that includes a plurality of microelectrodes. If the calculated value is within the acceptance range for test strips that have a plurality of microelectrodes, the user can proceed with the tests. However, if the calculated value is not within the acceptance range for test strips that have a plurality of microelectrodes, an appropriate error message is displayed on the test meter and the test meter will not allow the user to proceed With the tests.

Brief description of the drawings

A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description which sets forth illustrative embodiments, in which the principles of the invention are used, and the accompanying drawings of which:

Figure 1 is an exploded perspective view from above of an unmounted test strip in accordance with an exemplary embodiment of the present invention;

Figure 2 is a top view of the test strip as shown in Figure 1 after it has been mounted;

Figures 3 to 7 are top views of a distal part of a test strip partially assembled in accordance with exemplary embodiments of the present invention;

Figure 8 is a cross-sectional view of the test strip shown in Figure 3 through an arrangement of microelectrodes in a first working electrode according to an exemplary embodiment;

Figure 9 is a cross-sectional view through an arrangement of microelectrodes on a first working electrode of Figure 3 with additional layers coated on an insulating part that includes a reagent layer, adhesive pads, and a hydrophilic part The reagent layer is disposed on the distal side of the hydrophilic part according to an exemplary embodiment;

Figure 10 is a cross-sectional view through an arrangement of microelectrodes on a first working electrode of Figure 3 with additional layers coated on an insulating part that includes a reagent layer, adhesive pads, and a hydrophilic part The reagent layer is disposed above the insulation part according to an exemplary embodiment;

Figure 11 is a close-up view of the plurality of microelectrodes in the first working electrode of the test strip shown in Figure 3 in accordance with an exemplary embodiment;

Figure 12 is a top view of a test meter connected to the test strip of Figures 1 and 2;

Figure 13 is a simplified schematic view of the test meter of Figure 12 forming an electrical connection with the test strip of Figures 1 and 2;

Figures 14 and 15 are graphical representations of a test voltage applied to a working electrode of a test strip according to the methods of the present invention;

Figure 16 is a graphical representation of a current as a function of time (ie, a current-time transient) for a sample of fluid that is generated by a procedure;

Figure 17 is a graphic representation of

 as a function of

 which is generated by a procedure;

Fig. 18 is a flow chart illustrating a sequence of steps in a procedure for determining a hematocrit corrected analyte concentration reported by a test meter according to an exemplary embodiment;

Figure 19 is a flow chart illustrating a sequence of steps in a procedure for determining whether a body fluid or a control solution has been added to a test strip according to an exemplary embodiment;

Fig. 20 is a flow chart illustrating a sequence of steps in a process for determining whether a test strip includes a microelectrode arrangement according to an exemplary embodiment;

Figure 21 is a graphical representation illustrating simulated data that is used to differentiate between a test strip that includes a microelectrode arrangement and a test strip that does not include a microelectrode arrangement, in which the graphic representation is generated by A procedure; Y

Figures 22 and 23 are perspective and side views, respectively, of a medical device that is suitable for use in the present invention.

Detailed description of the exemplary embodiments

The disclosure below describes the measurement of a glucose concentration in a whole blood sample; However, the expert will recognize that the description is easily adapted to measure the properties of other analytes, such as cholesterol, ketone bodies or alcohol, and other fluids such as saliva, urine, interstitial fluid, or control solutions of the test strip. .

It is further understood that the present invention is not limited only to hematocrit correction and can also be applied for the correction of the effect of a variable viscosity or oxygen content in fluid samples. For example, complete blood samples may have a high viscosity for a variety of other reasons in addition to high hematocrit which includes a low temperature (for example, about 10 ° C), a high lipid concentration, and / or a concentration high protein

It will be further understood that the invention would also be applicable to reduce the effects to which oxygen results and / or the viscosity of physiological fluids other than blood. For example, physiological fluids may also include plasma, serum, interstitial fluid, and a combination thereof. It should be noted that it is not uncommon for the samples of interstitial fluid removed to be partially mixed with blood.

The sections will describe a test strip embodiment that can be used with an algorithm according to an embodiment to calculate a hematocrit corrected glucose concentration. Figure 1 is an exploded perspective view of a test strip 100, which includes multiple layers that are arranged on a substrate 5. These layers may include a conductive layer 50, an insulation layer 16, a reagent layer 22 , a layer 60 of adhesive, a hydrophilic layer 70, and a layer 80 above. The test strip 100 can be manufactured in a series of steps in which the conductive layer 50, the insulation layer 16, the reagent layer 22 and the adhesive layer 60 are deposited sequentially on the substrate 5 using, for example , a screen printing process as described in the publication prior to the granting of the United States under US20050096409A1 and the international applications published under Nos. WO2004040948A1, WO2004040290A1, WO2004040287A1, WO2004040285A2, WO2004040005A1, WO2004039897392, WO2004039897A2, In an alternate embodiment, an inkjet process can be used to deposit a reagent layer 22 on the substrate 5. An example of an inkjet process is described in US Pat.

No. 6,179,979. The hydrophilic layer 70 and the top layer 80 may be disposed from a roll material and laminated on the substrate 5. The test strip 100 also includes a distal part 3 and a proximal part 4 as shown in Figures 1 and 2.

The fully assembled test strip 100, as shown in Figure 2, includes an inlet 82 through which a blood sample can be introduced into a sample receiving chamber 84. The inlet 82 can be formed by cutting through a distal part 3 of the test strip 100. A blood sample 94 can be applied to the inlet 82 to fill a sample receiving chamber 84 such that glucose can be measured, as shown in Figure 12. Each of the side edges of a first pad 24 of adhesive and a second adhesive pad 26 that are adjacent to the reagent layer 22 defines a wall of the sample receiving chamber 84. A bottom or "floor" part of the sample receiving chamber 84 includes a substrate part 5, the conductive layer 50, and the insulation layer 16. An upper part or "roof" of the sample receiving chamber 84 includes a distal hydrophilic part 32.

For the test strip 100, as shown in Figure 1, the conductive layer 50 includes a reference electrode 10, a first working electrode 12, a second working electrode 14, a first contact 13, a second contact 15 , a reference contact 11, and a strip detection bar 17. Suitable materials that can be used for the conductive layer are Au, Pd, Ir, Pt, Rh, stainless steel, doped tin oxide, carbon, and the like. In one embodiment, the material for the conductive layer may be a carbon ink such as those described in US Patent 5,653,918. In another embodiment, the material for the conductive layer may be a metal metallized by ionic bombardment such as gold or palladium. A laser ablation pattern can be formed in the metal layer metallized by ionic bombardment to form a plurality of electrodes.

For the test strip 100, the insulation layer 16 includes a first opening 18 that exposes a part of the reference electrode 10, holes 20 exposing a part of the first working electrode 12, and a second opening 21 exposing a part of the second working electrode 14. The parts of the reference electrode 10, the first working electrode 12 and the second working electrode 14 that are exposed by the first opening 18, the holes 20 and the second opening 21, respectively, can be wetted by a sample of liquid such as shown in Figure 1. The holes 20 in the insulation layer expose a plurality of microelectrodes 120 as will be described with reference to Figures 5 to 9. In an exemplary embodiment, the insulation layer 16 is ink Ercon E6110–116 Jet Black Insulayer ™ that can be purchased through Ercon, Inc (Waltham, Massachusetts).

The reagent layer 22 may be disposed in a conductive layer portion 50, the substrate 5, and the isolation layer 16 as shown in Figure 1. In one embodiment, the reagent layer 22 may include an enzyme, a mediator which reacts selectively with glucose and a buffer to maintain a desired pH. Examples of enzymes suitable for use in the present invention may include either glucose oxidase or glucose dehydrogenase. More specifically, glucose dehydrogenase may have a pyrroloquinolin-quinone cofactor (referred to as abbreviated as PQQ or its common name which is methoxatin). Examples of a mediator suitable for use in the present invention may include either ruthenium ferricyanide or hexamine (Rum (NH3) 6). During the reactions as shown in equations 1 and 2, a proportionally reduced amount of mediator can be generated that is measured electrochemically to calculate a glucose concentration. Examples of buffers suitable for use in the present invention may include phosphate, citrate or citraconate. Examples of reagent formulations or inks suitable for use in the present invention can be found in U.S. Patents 5,708,247 and 6,046,051 and in published international applications WO01 / 67099 and WO01 / 73124.

In one embodiment, the formulation may include a 200 millimolar phosphate buffer having a pH of about 7 and a ruthenium hexamine mediator. The pH of about 7 was chosen because glucose oxidase has a sufficiently high activity at this pH when ruthenium hexamine is used as a mediator. In one embodiment, the formulation may have an enzymatic activity ranging from about 1,500 units / ml to approximately 8,000 units / ml. The range of enzymatic activity can be selected such that the glucose current does not depend on the level of enzymatic activity in the formulation provided that the level of enzymatic activity is within the level set forth above. The enzymatic activity should be large enough to ensure that the resulting glucose current will not be a function of small variations in the enzymatic activity. For example, the glucose stream will depend on the amount of enzyme activity in the formulation if the enzyme activity is less than 1,500 units / ml. On the other hand, for levels of enzyme activity higher than

8,000 units / ml, solubility problems may arise in which glucose oxidase cannot dissolve sufficiently in the formulation. Glucose oxidase can be purchased commercially through Biozyme Laboratories International Limited (San Diego, California, United States of America). Glucose oxidase can have an enzymatic activity of approximately 250 units / mg in which the units of enzymatic activity are based on an o-dianisidine assay with a pH of 7 and 25 ° C.

Optionally, the reagent layer 22 includes a matrix material that helps retain the reagent layer 22 on the conductive layer surface 50 in the presence of the fluid sample. Useful matrix materials include silica such as Cab-o-Sil® TS630 or Cab-o-Sil® 530 (Cabot Corporation, Boston, United States of America). Although it is not desired to be limited by any particular theory, it is believed that silica forms a gel network in the presence of the sample that effectively maintains the coating on the electrode surface. Other useful matrix materials include polymeric materials such as polyethersulfones, acrylic and methacrylic acid polymers; polymers obtained from starch, cellulose and other natural polysaccharides; polyamides and collagen. An example of a useful coating composition is disclosed in example 1 of US Pat. No. 5,708,247. The reagent layer 22 may also optionally include at least one stabilizing agent such as albumin, sucrose, trehalose, mannitol or lactose, an agent such as hydroxyethyl cellulose for adjusting viscosity, an antifoaming agent such as DC1500, and at least one wetting agent such as polyvinyl pyrrilidone or polyvinyl acetate.

For the test strip 100, the adhesive layer 60 includes the first adhesive pad 24, the second adhesive pad 26, and the third adhesive pad 28 as shown in Figure 1. In one embodiment, the layer 60 The adhesive may comprise a water-based acrylic copolymer pressure sensitive adhesive, which is commercially available through Tape Specialties LTD (Tring, Herts, UK; part no. A6435). The adhesive layer 60 is disposed in a portion of the insulation layer 16, the conductive layer 50, and the substrate 5. The adhesive layer 60 joins the hydrophilic layer 70 to the test strip 100.

The hydrophilic layer 70 includes a distal hydrophilic portion 32 and a hydrophilic proximal portion 34. As a non-limiting example, the hydrophilic layer 70 is a polyester having a hydrophilic surface such as an anti-fog coating that is commercially available through 3M.

For the test strip 100, the top layer 80 includes a transparent part 36 and an opaque part 38 as shown in Figure 1. The top layer 80 is arranged in and adhered to the hydrophilic layer 70. In one embodiment, the top layer 80 is a polyester. It should be noted that the transparent part 36 substantially overlaps the distal hydrophilic part 32 allowing a user to visually confirm that the sample reception chamber 84 has been sufficiently filled. The opaque part 38 helps the user to observe a high degree of contrast between a colored fluid such as, for example, blood inside the sample receiving chamber 84 and the opaque part 38 of the upper layer 80.

Figure 3 shows a simplified top view of a partial assembly of a test strip 100 that includes a first working electrode 12 in the form of a microelectrode arrangement 110 according to an exemplary embodiment. In general, the arrangement 110 of microelectrodes will increase the effects of radial diffusion resulting in an increase in the measured current density (the current per unit area of the

working electrode). Radial diffusion refers to the flow of the reduced mediator that diffuses to the first working electrode 106 in a non-perpendicular manner with respect to a plane of the first working electrode 106. In contrast, flat diffusion refers to the flow of the reduced mediator that diffuses to the first working electrode 106 in an approximately perpendicular manner with respect to a plane of the first working electrode 106. As a result of the increased radial diffusion, the application of a limiting test voltage for the microelectrode arrangement 110 may result in a test current achieving a non-zero steady state value that is independent of time. On the contrary, the application of a limiting test voltage to one other than a microelectrode will result in a test current approaching zero as time continues. Because the steady state value is independent of the time for a microelectrode arrangement 110, an effective diffusion coefficient D of the mediator in the blood sample can be calculated. In turn, the effective diffusion coefficient D can be used as an input to an algorithm to reduce the effects of hematocrit.

Referring again to Fig. 3, a distal portion 102 of the test strip 100 includes a reference electrode 10, a first working electrode 12 and a second working electrode 14. The first working electrode 10 is in the form of a microelectrode arrangement 110 that includes a plurality of microelectrodes

120.

In another embodiment shown in Figure 4, a distal portion 202 of a test strip 200 includes a reference electrode 204, a first working electrode 206 and a second working electrode 208. The test strip 200 differs from the test strip 100 in that a second working electrode 208 includes a microelectrode arrangement 210 with a plurality of microelectrodes 220. In yet another embodiment shown in Figure 5, both a first electrode Working 306 as a second working electrode 308 includes an arrangement of microelectrodes 310 with a plurality of microelectrodes 320.

Another embodiment of a test strip 400 having a microelectrode array 410 is shown in the figure.

6. A distal portion 402 of the test strip 400 includes a reference electrode 404, a working electrode 406 and a filling detector electrode 412. The working electrode 406 is in the form of a microelectrode arrangement 410 that includes a plurality of microelectrodes 420. The test strip 400 differs from the test strip 100 in that the working electrode 406 is upstream of the electrode 404 reference and does not include a second working electrode. As shown in Figure 6, the working electrode 406 may optionally have at least twice the surface area than the reference electrode 404.

Figure 7 shows yet another embodiment of a test strip 500 in which the test strip 500 includes a reference electrode 504 and a working electrode 506. In the present embodiment, the reference electrode 504 and the working electrode 506 have an approximately equal surface area. The working electrode 506 includes a microelectrode arrangement 510 and is downstream with respect to the reference electrode 504 with respect to the fluid flow in the test strip 500. Figure 7 also shows an optional filling detector electrode 512.

Many of the layers of test strip 100, as shown in Figure 1, can be used for test strips 100, 200, 300, 400 and 500, such as insulation layer 16, reagent layer 22 , the adhesive layer 60, the hydrophilic layer 70, and the upper layer 80.

Figure 8 is a cross-sectional view through the microelectrode arrangement 110 in the first working electrode 106 of Figure 3 showing that an isolation part 130 is disposed in the first working electrode 106. In an exemplary embodiment, the insulation portion 130 is contiguous with the insulation layer 16 of Figure 1. Therefore, in the present embodiment, the insulation portion 130 is printed at the same stage as the printing of Layer 16 insulation. The realization of a laser ablation of the insulation part 130 to form the holes 20 that expose a plurality of microelectrodes 120 can then form the microelectrode arrangement 110.

In another embodiment, the insulation part 130 is a separate element of the insulation layer 16 of Figure 1. In the present embodiment, the insulation part 130 is disposed on the first working electrode 106 at a separate stage of printing of insulation layer 16. The insulation part 130 may be disposed above and attached to the first working electrode 106 by processes such as ultrasonic welding, screen printing, or through the use of an adhesive. In the present embodiment, the holes 20 in the isolation part 130 may be formed before or after the insulation part 130 adheres to the first working electrode 106.

Figures 9 and 10 are cross-sectional views through microelectrode arrangement 110 on the first working electrode 106 of Figure 3 with additional layers including reagent layer 22, adhesive pads 24 and 26, and the hydrophilic part 32. The reagent layer 22 may be disposed in the distal hydrophilic part 32 as shown in Figure 9. In the present embodiment, the reagent layer 22 is very soluble, which ensures that when a sample of fluid is applied to the strip 100, the reagent layer 22 dissolves easily and the mediator rapidly diffuses to the microelectrodes 120. Alternatively, the reagent layer 22 may be disposed above an insulating portion 130 as shown in Figure 10. In the

In this embodiment, reagent layer 22 may be insoluble due to the immediate proximity of reagent layer 22 to microelectrodes 120. First and second adhesive pads 24 and 26 are applied in a manner such as to define a separation height L between the insulator part 130 and the reagent layer 22 (see Figure 9) or between the reagent layer 22 and the distal hydrophilic part (see Figure 10). The first and second adhesive pads 24 and 26 also define a width W of the reagent layer 22.

In order for the microelectrode arrangement 110 to have an increased effect due to radial diffusion, the insulation part 130 should have the appropriate dimensions. In one aspect, the insulation portion 130 may include a height H that is between about 1 micrometer and about 6 micrometers. It is necessary that the insulation part 130 be thin enough to allow radial diffusion. If the insulation part 130 is much larger than 6 micrometers, then the insulation part 130 interferes with the radial diffusion and would really encourage flat diffusion.

In another aspect shown in Figure 11, each microelectrode 120 should be separated far enough from the others to prevent a first microelectrode from competing with a second adjacent microelectrode for an oxidation mediator. Each microelectrode 120 can be separated a distance B that varies from about 5 times to about 10 times the diameter of the microelectrode 120. In an embodiment as shown in Figure 10, each microelectrode 120 can be evenly spaced along the entire length of the entire insulation part 130, in which a microelectrode can have six adjacent microelectrodes that adopt a hexagonal shape.

In yet another aspect, each microelectrode 120 should be small enough such that the proportion of the test current that is attributed to the radial diffusion is larger than the proportion of the test current that is attributed to the flat diffusion . The microelectrode 120 may have a disk shape with a diameter A ranging from about 3 micrometers to about 20 micrometers. In alternative embodiments, the microelectrode 120 may be in the form of a square, a rectangle, an ellipse or an oval.

In another aspect, the microelectrode arrangement 110 may be any geometric shape which includes, but is not limited to, a circle, an oval, a square or a rectangle. If they have a rectangular shape, the surface area is generally between about 0.3 and about 3 square micrometers.

Figure 12 illustrates a test meter 600 suitable for connection to the test strip 100. The test meter 600 includes a display 602, a housing 604, a plurality of user interface buttons 606, and a strip hole connector 608. The test meter 600 further includes an electronic circuit assembly inside the housing 604 such as a memory 620, a microprocessor 622, electronic components for applying a test voltage, and also for measuring a plurality of current values of proof. The proximal part 4 of the test strip 100 can be inserted into the strip hole connector 608. The display 102 may output a glucose concentration and be used to display a user interface to urge a user about how to perform a test. The plurality of user interface buttons 606 allows a user to operate the test meter 600 by browsing through the user interface software.

Figure 13 shows a simplified schematic diagram of a test meter 600 that works in conjunction with the test strip 100. The test meter 600 includes a first connector 612, a second connector 614, and a reference connector 610 that respectively form an electrical connection with the first contact 13, the second contact 15, and the reference contact 11. The three connectors mentioned above are part of the strip hole connector 608. When a test is performed, a first test voltage source 616 may apply a first test voltage V1 between the first working electrode 12 and the reference electrode 10. As a result of the first test voltage V1, the test meter 600 can then measure a first test current I1. Similarly, the second test voltage source 618 applies a second test voltage V2 between the second working electrode 14 and the reference electrode 10. As a result of the second test voltage V2, the test meter 600 can then measure a second test current I2. In one embodiment, the first test voltage V1 and the second test voltage V2 can be approximately equal, which allows a glucose measurement to be performed twice, in which a first measurement is made with the first working electrode 12 and a second measurement is made with the second working electrode 14. The use of two glucose measurements can increase accuracy by averaging the two results with each other. To simplify the description of the following sections, the algorithms for determining a glucose concentration corrected with hematocrit will be described for only one working electrode and the reference electrode. It should be apparent to one skilled in the art that the invention should not be limited to the working electrode and the reference electrode, but that multiple working electrodes can also be applied to the present invention.

The following procedures will describe algorithms that can be applied to microelectrodes and, more particularly, to microelectrode arrangements, in which the test current achieves a steady state value due to a higher radial diffusion rate.

Fig. 18 is a flow chart illustrating a sequence of steps in a method 700 that uses test meter 600 to apply a hematocrit correction at a glucose concentration according to

an exemplary embodiment.

The method 700 includes providing a test strip 100 with a reference electrode 10, a first working electrode 12, an optional second electrode 14 and a test meter 600, as set forth by step 710. The first electrode 12 Working includes a plurality of microelectrodes 120 (i.e., arrangement 110 of microelectrodes) each disk-shaped microelectrode having a diameter of about 3 micrometers to about 50 micrometers and being separated by about 5 to about 10 times the diameter thereof. . The reference electrode 10 includes a surface area that is at least equal to the surface area of the microelectrode arrangement 110.

Figure 14 is a graphical representation of a test voltage that is applied to the test strip 100 according to the procedure. Before a fluid sample is applied to the test strip 100, the test meter 600 is in a fluid detection mode in which a test voltage (not shown) of about 100 millivolts to about 600 millivolts, typically 400 millivolts is applied between the first working electrode 12 and the reference electrode 10. As set forth in step 720, the fluid sample is applied to test strip 100 at t0 and allowed to react with reagent layer 22 during a tR reaction period. The presence of a sample in the reaction zone of the test strip 100 is determined by measuring the current flowing through the first working electrode 12. It is determined that the start of the reaction period tR is initiated when the current flowing through the first working electrode 12 reaches a desired value, typically of about 0.125 nanoamperes (which is not shown), at which point a voltage of Zero millivolt test is applied between the first working electrode 12 and the reference electrode 10. The reaction period tR is typically between about 2 and about 3 seconds and is more typically about 2.5 seconds. After the reaction period tR, the test voltage in the exemplary procedure is applied to the test strip 100 at t1 for a total test time tT. In an alternative procedure shown in Figure 15, the reaction period tR is omitted, so that the start of the test begins as soon as sufficient current flows through the first working electrode 12.

As discussed in step 730, a limiting test voltage of about 100 millivolts to about 600 millivolts, typically 400 millivolts, is applied between the reference electrode 10 and the first working electrode 12 and the current is measured Test as a function of time, as illustrated in Figure 16. Note that the test current approaches a steady state value of current as time continues. An ISS steady state current value is measured when an equilibrium current value is obtained, as set forth by step 740. The ISS steady state current value is generally reached between about 0.5 and about 2 seconds after the test voltage is applied to the test strip 100.

As stated in step 750, the proportion of the test current with respect to a value of

steady state current

 It is calculated below for each time point at which the test current is measured. For the microelectrode arrangement 110 having a plurality of disk-shaped microelectrodes 120 in which a limiting test voltage is applied, the following equation estimates a proportion of the test current with respect to the steady state current value:

In which:

I (t) is the test current in microamps, measured at time t; ISS is the steady state value of current in microamps; rd is the radius of the microelectrode 120 in centimeters; t is the time in seconds; Y D is the effective diffusion coefficient in units of centimeter2 / second.

The effective diffusion coefficient D takes into account the diffusion of the mediator in a blood sample that has a dissolved reagent layer. In general, the effective diffusion coefficient D should decrease with the increase in hematocrit levels. Therefore, the effective diffusion coefficient D is a function of the hematocrit level and can be used in an algorithm to decrease the effects of hematocrit. The following will describe how to calculate the effective diffusion coefficient D and then apply the effective diffusion coefficient D to calculate a glucose concentration.

Using equation 4, the effective diffusion coefficient D can be calculated by plotting the values

 on the y axis and

 on the X axis as illustrated in Figure 17 and which has been set out in step 760. The resulting slope from the linear part of the line can be calculated and converted below and into the effective diffusion coefficient D, such as discussed in step 770. In practice, all the calculations that are required to determine the effective diffusion coefficient D would be previously programmed in the test meter as an algorithm. An advantage of this approach of determining the effective diffusion coefficient D is that the effective diffusion coefficient D is independent of the glucose concentration.

As discussed in step 780, the effective diffusion coefficient D can be used with equation 5 a

4-).

then to estimate the concentration of reduced mediator Cred (for example, the concentration of Fe (CN) 6

In which:

n is the number of electrons that are exchanged per molecule of reduced mediator; F is the Faraday constant.

Cred can then be used to estimate the glucose concentration corrected with hematocrit. For example, a calibration curve can be generated, in which the Y axis is Cred in which Cred is calculated for whole blood samples with a range of glucose and hematocrit concentrations. The X axis is the Gref reference glucose concentration of the same whole blood samples as measured in a reference glucose analyzer. The calibration intersection point can be subtracted from Cred following a division that uses a calibration slope to deparate the Gref glucose concentration. In summary, Equations 1 and 2 allow glucose concentrations to be calculated with a reduced effect from hematocrit when microelectrode arrangements are used as illustrated in Figures 3 to 7, which thus results in a more precise concentration of glucose.

Finally, the analyte concentration corrected with hematocrit is displayed on the test meter 600, as set forth in step 790.

Fig. 19 is a flow chart illustrating a sequence of steps in a procedure 800 that is used by the test meter 600 to establish whether a fluid sample is a body fluid (e.g., whole blood)

or a control solution according to an exemplary embodiment. A control solution is used to ensure that the test meter and the test strip are functioning properly. In one embodiment, analyte concentrations for a body fluid are averaged over a period of time to assess the patient's health. If a control value solution is stored in the test meter as an analyte concentration, the average analyte concentration for body fluid will be incorrect. Therefore, it is advantageous to have a procedure to distinguish between a body fluid and a control solution.

The method 800 includes providing a test strip 100 with a reference electrode 10, a first working electrode 12, an optional second electrode 14 and a test meter 600, as set forth by step 810. The first electrode 12 Working includes a plurality of microelectrodes 120 (i.e., arrangement 110 of microelectrodes) each disk-shaped microelectrode having a diameter of about 3 to about 50 micrometers and being separated by about 5 to about 10 times the diameter thereof. . The reference electrode 10 includes a surface area that is at least equal to the surface area of the microelectrode arrangement 110.

As discussed in step 820, the fluid sample is applied to the test strip 100 at t0 and allowed to react with the reagent layer 22 during a reaction period tR (see Figure 14). The presence of a sample in the reaction zone of the test strip 100 is determined by measuring the current flowing through the first working electrode 12. The start of the reaction period tR is determined to be initiated when the current flowing through the first working electrode 12 reaches a desired value, typically of approximately 0.125 nanoamperes (which is not shown), at which point a voltage of test between approximately - 50 millivolts and approximately + 50 millivolts, typically approximately zero millivolts, is applied between the first working electrode 12 and the reference electrode 10. The reaction period tR is typically between about 2 and about 3 seconds and is more typically about 2.5 seconds. After the reaction period tR, the test voltage in the exemplary procedure is applied to the test strip 100 at t1 for a total test time tT. In an alternative procedure shown in Figure 15, the reaction period tR is omitted, so that the start of the test begins as soon as sufficient current flows through the first working electrode 12.

As discussed in step 830, a limiting test voltage of approximately 100 millivolts

Up to approximately 600 millivolts, typically 400 millivolts, is applied between the reference electrode 10 and the first working electrode 12 and the test current is measured as a function of time, as illustrated in Figure 16. A value of ISS steady state current is measured when an equilibrium current value is obtained, as set forth by step 840. The ISS steady state current value is generally reached between about 0.5 and about 2 seconds after that the test voltage is applied to the test strip 100.

As stated in step 850, the proportion of the test current with respect to a value of

steady state current

 It is calculated below for each time point at which the test current is measured. For the microelectrode arrangement 110 having a plurality of disk-shaped microelectrodes 120 in which a limiting test voltage is applied, equation 4 above is used to estimate a proportion of the test current with respect to the status value stationary current.

Using equation 4, the effective diffusion coefficient D can also be calculated by plotting the values

in

the y axis and

 on the X axis as illustrated in Figure 17 and which has been exposed in step 860. The resulting slope from the linear part of the line can be calculated and converted below and into the effective diffusion coefficient D, such as discussed in step 870.

As discussed in step 880, the effective diffusion coefficient D can be used with equation 5 above to estimate the concentration of reduced mediator Cred (eg, the concentration of Fe (CN) 6 4–).

To determine the type of fluid sample (e.g., body fluid or control solution) that is applied to the test strip 100, the test meter 600 compares a measured value for the effective diffusion coefficient D with respect to an acceptance interval for the body fluid and an acceptance interval for the control solution, as set forth by step 880. For whole blood with a hematocrit level of between about 20% and about 70%, the Effective diffusion coefficient D is typically between about 0.7 x 10 –6 centimeters2 / second and about 2.7 x 10 –6 centimeters2 / second. The effective diffusion coefficient D for the control solution is typically between about 4.0 x 10 -6 centimeters2 / second and about 7.2 x 10 -6 centimeters2 / second.

Finally, the test meter 600 displays an appropriate error message if the fluid sample is not in the acceptance range for the body fluid or the control solution, depending on what type of fluid sample has been applied to the strip 100 test, or allows the user to proceed with the tests, as set forth by step 890.

The estimated effective diffusion coefficient D can also be used to distinguish between test strips 100 that include microelectrodes 120 and those that do not include microelectrodes 120. The estimated effective diffusion coefficient d can also be used to determine whether reagent layer 22 It has been formulated or coated incorrectly. Fig. 20 is a flow chart illustrating a sequence of steps in a process 900 that is used by a test meter to determine if a test strip 100 includes a plurality of microelectrodes 120 and includes a reagent layer 22 that has been Correctly formulated and coated according to an exemplary embodiment.

The method 900 includes providing a test strip 100 with a reference electrode 10, a first working electrode 12, an optional second electrode 14 and a test meter 600, as set forth by step 910. The first electrode 12 Working includes a plurality of microelectrodes 120 (ie, an arrangement 110 of microelectrodes), each microelectrode 120 having a diameter between about 3 micrometers and about 50 micrometers and being separated by about 5 to about 10 times the diameter thereof. The reference electrode 10 includes a surface area that is at least equal to the surface area of the microelectrode arrangement 110.

As set forth in step 920, the fluid sample is applied to the test strip 100 at t0 and allowed to react with the reagent layer 22 during a reaction period tR (see Figure 14). The presence of a sample in the reaction zone of the test strip 100 is determined by measuring the current flowing through the first working electrode 12. It is determined that the start of the reaction period tR is initiated when the current flowing through the first working electrode 12 reaches a desired value, typically of about 0.125 nanoamperes (which is not shown), at which point a voltage of Zero millivolt test is applied between the first working electrode 12 and the reference electrode 10. The reaction period tR is typically between about 2 and about 3 seconds and is more typically about 2.5 seconds.

After the reaction period tR, the test voltage in the exemplary procedure is applied to the test strip 100 at t1 for a total test time tT. In an alternative procedure shown in Figure 15, the reaction period tR is omitted, so that the start of the test begins as soon as sufficient current flows through the first working electrode 12.

As discussed in step 930, a limiting test voltage of about 100 millivolts to about 600 millivolts, typically 400 millivolts, is applied between the reference electrode 10 and the first working electrode 12 and the current is measured test as a function of time, as illustrated in Figure 16. An ISS steady state current value is measured when an equilibrium current value is obtained, as set forth by step 940. The value of ISS steady state current is generally reached between about 0.5 and about 2 seconds after the test voltage is applied to the test strip 100.

As stated in step 950, the proportion of the test current with respect to a value of

steady state current

It is calculated below for each time point at which the test current is measured. For the microelectrode arrangement 110 having a plurality of disk-shaped microelectrodes 120 in which a limiting test voltage is applied, equation 4 above is used to estimate a proportion of the test current with respect to the status value stationary current.

Using equation 4, the effective diffusion coefficient D can be calculated by plotting the values

 on the y axis and

 on the X axis as illustrated in Figure 17 and which has been set out in step 960. The resulting slope from the linear part of the line can be calculated and converted below and into the effective diffusion coefficient D, such as stated in step 970.

Next, as set forth by step 980, a temperature corrected diffusion coefficient

 It is calculated by substituting effective diffusion coefficient D in equation 6 below that approximates diffusion as a function of temperature in a gel.

In which:

 is the effective diffusion coefficient corrected in temperature in centimeter2 / second; D is the estimated effective diffusion coefficient in centimeter2 / second; 8 is a known constant for diffusion as a function of temperature; T is the Kelvin temperature of the fluid sample as measured by the test meter and in overall it is between about 283 K and about 317 K; Y T0 is a reference temperature (for example, room temperature) in Kelvin. T0 varies between approximately 293 Kelvin and approximately 298 Kelvin.

Next, the test meter 600 determines whether the test strip 100 includes a microelectrode array 110 and a reagent layer 22 formulated and coated correctly by comparing a calculated value

of effective diffusion coefficient corrected in temperature versus an acceptance interval, as it has been

exposed by step 990. The effective diffusion coefficient corrected in temperature

 it is typically about 1.8 x 10 –6 centimeters2 / second and is usually between about 1.6 x 10 –6 centimeters2 / second and 2.0 x 10 –6 centimeters2 / second.

Finally, if the value calculated for the effective diffusion coefficient corrected in temperature

 is within the acceptance interval, the user is allowed to proceed with the tests. Yes, however, the value

calculated for the effective diffusion coefficient corrected in temperature

 is outside the acceptance range, the test meter 600 displays an appropriate error message (for example, the test strip was not recognized) for the user, as set forth by step 995.

Figure 21 is a graphical representation illustrating simulated values for the effective diffusion coefficient

corrected in temperature

 for test strips 100 that include a microelectrode arrangement 110 and those that do not include a microelectrode arrangement 110. Arrowhead M indicates an appropriate range of

values for the effective diffusion coefficient corrected in temperature

 for test strip 100 which includes 5 an arrangement 110 of  microelectrodes The values for the effective diffusion coefficient corrected in

temperature

 of the test strips that do not include a microelectrode arrangement 110 would be outside the arrowhead M as indicated by the arrowheads N1 and N2 in this graphic representation.

Figures 22 and 23 are perspective and side views, respectively, of an integrated medical device 1000 that may include a plurality of microelectrodes 120 in accordance with the exemplary embodiments. The integrated medical device 1000 includes a test strip 1004 and a skin penetration element 1002. The test strip 1004 has a reaction area 1005 which is composed of the reagent layer 22 coated on the reference electrode and one or more working electrodes (not shown). The electrical contacts 1006 appear at a proximal end 1010 of the integrated medical device 1000 and are formed by any suitable conductive material, such as gold, silver, platinum or carbon. The skin penetration element 1002 includes a lancet 1020 adapted to pierce a user's skin and draw blood into the reaction area 1005. The dermal tissue penetration element 1002 is adhered to the test strip 1004 by a layer 1014 of adhesive. This adhesive layer 1014 may be a pressure sensitive or heat sealed adhesive. The lancet 1020 includes a lancet base 1022 that terminates at the distal end 1012 of the mounted test strip. Additional descriptions of the integrated medical devices that can be used with the

The present invention is found in the United States Patent Application No. 10 / 432,827 (published as 2004/0096959 on May 20, 2004) and in the United States Patent Application No. 10 / 143,399 (published as 2003/0143113 on July 31, 2003).

Claims (15)

one.

 A method of calculating a glucose concentration corrected with hematocrit in a fluid sample, the procedure comprising:

providing a test strip comprising a reference electrode and a working electrode formed with a plurality of microelectrodes and coated with a reagent layer; apply a sample of fluid to the test strip during a reaction period; apply a test voltage between the reference electrode and the working electrode; measure a test current as a function of time; measure a steady state current value when the test current has reached equilibrium; calculate a proportion of the test current with respect to the steady state current value; plot the proportion of the test current with respect to the steady state current value as a function of the inverse of the square root of time; calculate an effective diffusion coefficient from the slope of the regression graphical representation linear of the proportion of the test current with respect to the steady state current value as a function of the inverse of the square root of time; Y calculate a corrected concentration with analyte hematocrit.

2.

 A method of determining a type of fluid sample applied to the test strip, the procedure comprising:

provide a test strip that has a reference electrode and a working electrode, in which the Work electrode is formed with a plurality of microelectrodes and coated with a reagent layer; apply a sample of fluid to the test strip during a reaction period; apply a test voltage between the reference electrode and the working electrode; measure a test current as a function of time; measure a steady state current value when the test current has reached a Balance; calculate a proportion of the test current with respect to the steady state current value; plot the proportion of the test current with respect to the steady state current value as a function of the inverse of the square root of time; calculate an effective diffusion coefficient from the slope of the regression graphical representation linear of the proportion of the test current with respect to the steady state current value as a function of the inverse of the square root of time; determine a type of a fluid sample applied to the test strip by comparing a measured value for the effective diffusion coefficient against an acceptance interval for a body fluid and a control solution; Y display an appropriate error message if the effective diffusion coefficient does not exceed the interval of Acceptance for the type of fluid sample applied to the test strip.

3.

 The method of claim 1 or 2, wherein the step of calculating the effective diffusion coefficient uses an equation of the form:

in which: I (t) is the current value in microamps, measured at time t; ISS is the steady state current in microamps; rd is the radius of a microelectrode in centimeters; Y t is the time in seconds. 4. The method of claim 3, wherein the step of calculating a concentration corrected with Hematocrit of the analyte further comprises: replacing the estimated diffusion coefficient in an equation of the form: in which: Cred is a reduced mediator concentration; ISS is the steady state current in microamps; n is the number of electrons that are exchanged per ion that undergoes a reaction of oxidation / reduction; F is Faraday's constant; D is the estimated diffusion coefficient in centimeter2 / second; and rd is the radius of the microelectrode in centimeters; generate a calibration curve in which a Y axis is the reduced mediator concentration and an X axis is a concentration of reference analyte; calculate an analyte concentration by subtracting a calibration intersection point with respect to the Reduced mediator concentration and divide with a calibration slope.

5.

 The method of claim 1 or 2, wherein the test strip further comprises an insulation part that is disposed in the plurality of microelectrodes and the insulation part has a height between about one micrometer and six micrometers.

6.

 The method of claim 1 or 2, wherein the reference electrode and the working electrode are composed of gold.

7.

 The method of claim 1 or 2, wherein the reagent layer comprises an enzyme, a mediator and a buffering agent in which the mediator comprises ruthenium (III) hexamine.

8.

 The method of claim 1 or 2, wherein the diameter of each of the plurality of microelectrodes is between about 5 micrometers and 50 micrometers.

9.

 The method of claim 1 or 2, wherein each of the plurality of microelectrodes is separated by a distance ranging from about 5 to 10 times the diameter of a microelectrode or ranging from about 25 micrometers to 500 micrometers.

10.

 The method of claim 1 or 2, wherein the shape of each of the plurality of microelectrodes is selected from a group consisting essentially of a circle, a square, a rectangle, an oval and an ellipse and combinations thereof. .

eleven.

 A system that includes:

a test strip having a reference electrode and a working electrode, in which the working electrode is formed with a plurality of microelectrodes and is coated with a reagent layer, and a test meter comprising: an electronic circuit for applying a test voltage between the reference electrode and the working electrode; and a signal processor to measure a test current and to calculate an effective diffusion coefficient.

12.

 The system of claim 11, wherein the test strip further comprises an insulation part that is disposed in the plurality of microelectrodes and the insulation part has a height of between about one micrometer and six micrometers.

13.

 The system of claim 11, wherein the effective diffusion coefficient is calculated from the slope

of the graphical representation of linear regression based on an equation of the form: in which: I (t) is the test current in microamps, measured at time t; ISS is the steady state current in microamps; rd is the radius of a microelectrode in centimeters; Y t is the time in seconds. 14. The system of claim 13, wherein a hematocrit corrected glucose concentration is calculated in which: Cred is a reduced mediator concentration; ISS is the steady state current in microamps; n is the number of electrons that are exchanged per ion that undergoes a reaction of oxidation / reduction; 5 F is the Faraday constant; D is an estimated diffusion coefficient in centimeter2 / second; Y rd is the radius of the microelectrode in centimeters. 15. The system of claim 14, wherein an effective diffusion coefficient is used to discriminate between a sample of whole blood fluid and a sample of control solution fluid by comparing the coefficient of 10 effective diffusion with an acceptance interval for whole blood and an acceptance interval for the control solution and the effective diffusion coefficient is used to determine if a test strip is formed with a In which:  it is an effective diffusion coefficient corrected in temperature in centimeter2 / second; D is an estimated effective diffusion coefficient in centimeter2 / second; 8 is a known constant for diffusion as a function of temperature; T is the Kelvin temperature of the fluid sample as measured by the test meter; Y T0 is a reference temperature in Kelvin.